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The U.S. FDA (United States Food and Drug Administration), MHRA (Medicines and Healthcare products Regulatory Agency), HPRA (Health Products Regulatory Authority) and CFDA (China Food and Drug Administration), amongst others, routinely make observations about disinfectant validation studies and disinfectant practices. The U.S. FDA Guidance for Industry, Sterile Drug Products Produced by Aseptic Processing (Aseptic Processing Guide September 2004) states « Each manufacturer must have a formal program governing the qualification, use and disposal of disinfectants. » The current United States Pharmacopeia, USP 37 <1072> chapter gives some guidance on selection, use and qualification of disinfectants . There is no question that drug manufacturers should provide evidence that room decontamination programs achieve and maintain desired contamination control levels. This paper will provide considerations and discuss best practices for validating disinfectants used in drug manufacturing areas.
It is important to understand that disinfec- tant validation is a process that includes three distinct components. These components are: disinfectant qualification testing or in vitro studies, in situ evaluations, and environmental monitoring with trending during routine operation. In vitro (Latin for « in glass ») studies are those that are conducted in a laboratory or artificial environment. Because there are a number of variables that can impact disinfectant performance under actual use conditions, it is important to conduct in vitro studies to demonstrate that a particular product is inherently effective against a par- ticular organism under well-defined conditions, such as concentration and contact time. Most countries require in vitro testing in order to register and market a disinfectant or sporicidal product. The product labeling reflects the particular organisms (e.g. American Type and Culture Collection or ATCC strains) that were included in these studies, and the specific conditions under which testing was conducted (e.g. temperature, concentration, contact time, etc.). However, the testing required for product registration typically does not meet the needs of pharmaceutical manufacturers who must comply with regulatory expectations.
Regulatory expectations include:
Demonstrating effectiveness on materials of construction that are representative of actual manufacturing surfaces (e.g. epoxy flooring, LexanTM polycarbonate curtains, etc.), demonstrating effectiveness against environmental isolates, and demonstrating effectiveness when applied following the organization’s Standard Operating Procedures (SOPs) e.g. dilution water quality, wet contact time, expiration dating of use-solution, soil loading, application techniques, etc..
The condition and composition of the surface can have an adverse impact on the performance of the disinfectant for a number of reasons, e.g. reactivity to disinfectant, porosity, etc. Warning Letter January 29, 2013: « The coupons used in the « Disinfectant Efficacy Verification for Hard Surfaces…. »were not representative of the surfaces found in the tissue processing laboratories (TPL) and BioAdhesive laboratories. For example, ___ was used in the study to represent biological safety cabinets, laminar flow hoods, and tables in the processing and manufacturing areas. However, the equipment is comprised of ___ » « All surfaces that are used in critical processing and manufacturing areas were not evaluated… »
Environmental isolates are of particular interest simply because they were isolated from the manufacturing environment, which clearly indicates that they are being introduced into the facility with some degree of regularity and therefore, may pose a risk to the product. As far back as the early 1990’s, there has been an expectation that pharmaceutical manufacturers include environmental isolates in validation studies.
GMP TRENDS, November 15, 1993, « The Firm’s sanitizing agents have not been validated with environmental microorganisms which have been observed to be part of the firm’s environmental bioburden. »
SOPs should include specific details for preparation of the disinfectant (e.g. use-dilution concentration, water quality, water temperature, etc.), required wet contact time for the surface, application devices and instructions (e.g. mopping direction and room grid) and expiry dating of both the use-dilution and the opened source container of disinfectant or sporicide.
FDA Warning Letter October 31, 2008, « However your response to our FDA-483 is inadequate because the following were not addressed: Effectiveness of _____ solution at the dilution used, and 2) effectiveness of ________ throughout the shelf life (up to the expiry date). »
In vitro testing
When considering several potential disinfectants or sporicidal agents, it may be prudent to begin in vitro testing with suspension studies. A suspension study in its most simple form involves exposing a known inoculum of a specific organism to a known concentration of disinfectant or sporicide, for example, for a specified period of time. This type of assessment offers a relatively quick read on whether or not a particular product and/or set of use conditions (e.g. water quality, temperature) is effective against a particular organism or group of organisms. Once the suspension studies are complete, a comparison of effectiveness of various products should allow selection of a limited number of highly effective products that can then be included in more rigorous testing, including coupon studies representing the materials of construction (MOC) of areas or equipment to be treated.
A number of recent FDA Warning Letters have been focused on coupon studies. In particular, the regulators have expressed concern about the selection and condition of MOC failing to represent both the actual MOC and the condition of such materials in manufacturing areas. A recent FDA warning letter stated: « All surfaces that are used in critical processing and manufacturing areas were not evaluated. » (FDA Warning Letter January 29, 2013). « There is no evaluation of the effectiveness of cleaning and chemical agents used to control microbial populations on approximately 15 different hard surfaces (e.g. Aluminum) found in classified areas used to manufacture sterile products. » (GMP Trends November 1, 2013). When developing a testing matrix, it is important to consider MOC that fairly represent the manufacturing surfaces and that represent the condition of the surfaces. In an ideal world, damaged surfaces would be immediately repaired or replaced. However, this is not always possible, and if damaged surfaces are to be kept in use for an extended period of time (e.g. until the next scheduled maintenance event), then damaged surfaces must be represented in coupon studies. « The materials that were tested in the Disinfectant Efficacy study were not representative of all the surfaces present in the Aseptic Processing Area. » « The stainless steel coupons tested did not represent these damaged surfaces. » (Warning Letter May 25, 2011)
In addition to the MOC and condition of coupons, selection of environmental isolates to include in testing is a key consideration. Selection should include organisms most commonly isolated from manufacturing surfaces and personnel (e.g. gram positive and gram negative bacteria), organisms that are known to demonstrate resistance to decontamination or otherwise harsh conditions (e.g. spore-formers, mold), and organisms that are introduced into the area via known vectors, such as raw materials. In the event that a facility is newly operational and a substantial body of isolates has not yet been established, inclusion of a broad spectrum of organisms sourced from ATCC, for example, may be considered.
In addition to MOC and isolate selection, regulators will also scrutinize other aspects of the in vitro work including, log reduction goals and results, recovery and neutralization studies, and controls. A recent FDA Warning Letter stated: « Your disinfectant qualification for (b)(4) and (b)(4) bi-spore disinfectants documented that the log reduction criteria (Bacteria ≥ 4, Fungi ≥ 3) was not met when challenged with multiple organisms in a variety of surfaces. » (FDA Warning Letter, October 7, 2011). « There is no assurance that the disinfectant is effective against mold, since it did not meet your established recovery rate acceptance criterion in the December 2001 « Disinfectant Validation and Efficacy Study by the Surface Test Method » study. » (FDA Warning letter, May 24, 2007)
The study design and method used for in vitro testing of disinfectants by a pharmaceutical manufacturer must be carefully planned and be scientifically justifiable to the regulatory authorities. USP <1072> provides very little guidance on how these studies should be performed. While USP <1072> does refer to AOAC (Association of Official Analytical Chemists) methods, these are not necessarily appropriate when qualifying a disinfectant for use in a pharmaceutical facility and moreover, some AOAC tests, such as Use-Dilution Method require exceptional expertise as they are very technique dependent and often difficult to perform consistently. Unfortunately, there is not one perfect testing method. However, there are several published methods that do provide good general information for performing these studies and that can be modified and adapted for use in disinfectant qualification testing.
Such examples include, the ASTM E2197- 02(American Society for Testing and Materials) Quantitative Carrier Test (QCT) and the European Norm EN13697. These methods utilize stainless steel disks (other surfaces can be adapted) inoculated with the challenge microorganism that are treated with the disinfectant followed by neutralization and quantitation of survivors in order to establish the activity of the product.
In situ testing
In situ testing demonstrates that the disinfectant or sporicidal agent in conjunction with preparation procedures and application procedures used by the facility and employees are effective at maintaining the environmental microbial levels deemed necessary for production of the target product. Efficacy of the disinfection program is demonstrated through evaluation of environmental monitoring data both over time and during « worst-case » remediation events. For example, many firms will compare environmental data pre and post decontamination after a preventative maintenance shut-down, when the room is more likely to show relatively high levels of environmental contamination. It is critically important that the procedures used to decontaminate the area during the in situ evaluation reflect the written SOPs, as evidenced by regulatory feedback, « There is a lack of written procedures assigning responsibility, providing cleaning schedules, and describing in sufficient detail the method, equipment and materials to be used for sanitation. Specifically, your firm does not maintain written and approved procedures for the cleaning/disinfection of equipment and materials. » (FDA 483, June 11, 2013). Clearly, the personnel who are assigned to perform these functions, must have sufficient training and oversight. Failure to have and/or to follow written procedures, problems with cleaning, sanitization, and maintenance, and failure to provide sufficient training are amongst the most frequently occurring FDA 483 observations.
USP 1072 provides some general guidance for in situ testing, « To demonstrate the efficacy of a disinfectant within a pharmaceutical manufacturing environment, it may be necessary to conduct the following tests…a statistical comparison of the frequency of isolation and the numbers of microorganisms isolated prior to and after the implementation of a new disinfectant. » Further, the FDA Aseptic Processing Guide from 2004 states, « the effectiveness of these sanitization procedures should be measured by their ability to ensure that potential contaminants are adequately removed from surfaces (i.e., via obtaining samples before and after sanitization). » It is clear that evaluation of surfaces in order to compare contamination levels before and after sanitization or disinfectant treatment is an expectation in substantiating disinfectant performance.
Environmental monitoring and trending
Environmental monitoring practices, including frequency, location, and number of samples per sampling interval, should be based upon best available guidance documents and a valid scientific rationale suited to the type of product being manufactured. That being said, a single day of environmental monitoring data is but a snapshot in time, and cannot, alone, convey much useful intelligence about the state of control of a manufacturing area. Ongoing environmental monitoring, with data trending, is further validation that a holistic contamination control program is effective. It is recommended that any organisms detected be identified to the species level, and that they be stored for inclusion in future in vitro studies. Data should be reviewed periodically for negative trends; once a month is a common frequency. Additionally, criteria must be established for identifying a negative trend. « Procedures do not define how data must be presented in the (b) (4) trend reports generated by… The investigations include environmental data for the aseptic area that is reviewed for trends. However, there is no procedure that defines the search criteria for trending. No evaluation of environmental monitoring data for the support areas within the aseptic core were conducted during the investigations. » FDA 483 March 01 2013.
Disinfectant validation is a process that includes in vitro studies, where the disinfectant or sporicidal agent can be evaluated under highly controlled conditions; in situ evaluations which demonstrate how effective the disinfectant or sporicidal agent is under actual use conditions (typically conducted in a worst-case environment); and routine environmental monitoring with trending and assessment of negative trends. While there is no single regulatory or advisory document available that offers a blueprint for development of a disinfectant validation study, there are several documents and references, including FDA 483 observations and Warning Letters, which both highlight pitfalls and offer solid input on study design.
Jim POLARINE – STERIS CORPORATION
MHRA, Annex 1, Manufacture of Sterile Medicinal Products, Rules and Guidance for Pharmaceutical Manufacturers and Distributors. January 2 2014: 74-85.
European Standard, EN 13697. Chemical disinfectants and antiseptics – Quantitative non-porous surface test for the evaluation of bactericidal and/or fungicidal activity of chemical disinfectants used in food, industrial, domestic, and institutional areas – Test method and requirements without mechanical action (phase 2/step 2). August 2001.
FDA Inspections, Compliance, Enforcement and Criminal Investigations, Warning Letter WL 320-12-01, October 7, 2011. Available at :
FDA Inspections, Compliance, Enforcement and Criminal Investigations, Warning Letter WL13ATL-07,January 29, 2013.
Available at : http://www.fda.gov/iceci/enforcementactions/warningletters/2013/ucm341423.htm.
FDA Office of Global Regulatory Operations and Policy, 483 Observation, FEI Number 1021343 2/12/2013-3-01/2013. Available at:
FDA Inspections, Compliance, Enforcement and Criminal Investigations, Warning Letter WL October 7, 2011. Available at: www.fda.gov/iceci/enforcementactions/warningletters/2011/ucm275960.htm.
FDA Inspections, Compliance, Enforcement and Criminal Investigations, Warning Letter WL May 24, 2007. Available at: www.fda.gov/ICECI/EnforcementActions/WarningLetters/2007/ucm076398.htm
FDA Inspections, Compliance, Enforcement and Criminal Investigations, Warning Letter WL October 31, 2008. Available at: www.fda.gov/ICECI/EnforcementActions/WarningLetters/2008/ucm1048078.htm.
483 Observation, 11/15/93. GMP Trends, Inc. at: www.gmptrends.com.
483 Observation, 11/1/13. GMP Trends, Inc. at: www.gmptrends.com.
ASTM International, ASTM E2197-11 Standard Quantitative Disk Carrier Test Method for Determining The Bactericidal, Virucidal, Fungicidal, Mycobactericidal and Sporicidal Activities of Liquid Germicides. West Conshohocken, PA 2011.